We propose to use dynamic computational imaging, in vivo, to increase our understanding of how endothelial cells and their precursors are precisely patterned in space and time. Dynamic imaging will be combined with a new array of elegant molecular tools tailored for use in quail embryos. To accomplish our goals we propose: 1) To establish a dynamic conceptual framework of how primary vascular patterns emerge in warm-blooded animals;2) To prepare lineage-fate maps of angioblasts and their progeny and thereby define the primary spatiotemporal pattern of vascular-specific gene expression;3) To determine the function of key signaling molecules implicated in vascular cell-autonomous motility and differentiation;and 4) To develop biologically-grounded mathematical models and computer simulations of vasculogenesis in amniotes. The work will include preparation of position-fate and lineage maps of angioblasts employing fluorescent reporter proteins and transgenic quail expressing endothelial cell markers. Cell biological reagents and RNAi methods will be used to target signaling molecules that impact primary vascular pattern formation from its inception to its completion, i.e., HH Stages 1-10. The motion of surrounding ECM fibrils, including matrix-bound VEGF, and bulk tissue flow will be quantified and distinguished from the local cell-autonomous motility ("migration") of angioblasts and primordial endothelial cells, in vivo. We will then collaborate with physicists and mathematicians to construct novel, biologically-grounded, models of vasculogenesis, and also design computer simulations. The models and simulations will be based on both the empirical time-lapse imaging data, and the experimental perturbation/gene silencing data. The overall goal, therefore, is to "solve" all relevant cellular motion, tissue flow and ECM motion patterns that define vasculogenesis much like a physical biochemist solves the structures of a protein. Based on this conceptual framework we will then intervene experimentally at critical junctures to illuminate how key molecular mechanisms operate, in situ, in "real-time". During the lifetime of the proposed studies we will observe gene-silencing on-the-fly in a warm-blooded embryo. PUBLIC HEALTH RELEVANCE: The proposed work will help decipher the cell and tissue behavior required to form healthy vessels and to help explain the mechanisms underlying the failure of diseased vessels and the root causes of vascular malformations in fetuses and infants. A major strength of the application is that the work employs "real-time" motion analysis in live tissues;thus we are not using a model of vascularization on the contrary, we are directly studying the process in a real world biological setting. Knowledge gained by our dynamic computational studies on regulation of early vessel growth and pattern formation will, ipso facto, reveal underlying causes of vascular disease.